Biopolymers and biopolymer blends, and method for producing same

ABSTRACT

Biopolymers and biopolymer blends, including block copolymers, prepared via enzyme-mediated catalysis preferably in a microorganism host in which the reaction conditions are selected to produce biopolymers and biopolymer blends having particular chemical compositions and microstructures.

STATEMENT AS TO FEDERALLY SPONORERD RESEARCH

This invention was funded pursuant to a grant received from theConsortium for Plant Biotechnology Research (Grant No. OR22072-77).Accordingly, the government may have rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates to preparing biopolymers and biopolymer blends byenzyme-mediated catalysis e.g., in a microorganism host. Biopolymers arepolymers that can be prepared via enzyme-mediated catalysis either invitrn or in a microorganism host such as a bacterium. These biopolymersare desirable because they are biodegradable and biocompatible, makingthem suitable for a number of applications. In a typical procedure, oneor more nutrients is fed to bacterial cells containing polymeraseenzymes capable of processing the nutrients to form the desiredbiopolymer. The biopolymer is deposited in the form of osmoticallyinert, intracellular granules that are then extracted from the cells.This procedure has been used, for example, to prepare homopolymers andrandom copolymers containing 3-hydroxy organoate units.

SUMMARY OF THE INVENTION

The inventors have discovered how to control the microstructure andchemical composition of biopolymers and biopolymer blends produced viaenzyme-mediated catalysis. Accordingly, it is now possible to preparebiopolymers and biopolymer blends that are tailored to meet the needs ofa specific application. In particular, block copolymers andphase-separated blends can be prepared.

The phase-separated blends feature particles having abiopolymer-containing core substantially surrounded by abiopolymer-containing shell in which the core and shell have differentchemical compositions. The biopolymers may be in the form ofhomopolymers, copolymers, or combinations thereof, where “homopolymer”refers to a polymer having a single type of monomer unit and “copolymer”refers to a polymer having two or more different monomer units. Thethickness of the shell is less than about 1 micrometer, preferably lessthan about 0.1 micrometer, and more preferably less than about 0.05micrometer.

A number of different core/shell combinations can be prepared. Accordingto one embodiment, the core includes a homopolymer and the shellincludes a copolymer, preferably in which the copolymer and homopolymerhave common monomer units. Alternatively, the core can include thecopolymer and the shell can include the homopolymer. The particle canfurther include one or more additional shell layers.

Particularly useful biopolymers are homopolymers and copolymers thatinclude hydroxy organoate units. Such units have the general formula:

where R is a group containing between 1 and 30 carbon atoms, inclusive,and n is at least 1. Examples include 3-hydroxy organoate units (n=1)and 4-hydroxy organoate units (n=2). Specific examples include 3-hydroxybutyrate (R=methyl; n=1), 3-hydroxy valerate (R=ethyl; n=1), andcombinations thereof. In one embodiment, the core includes a homopolymercontaining 3-hydroxy butyrate units and the shell includes a copolymercontaining 3-hydroxy butyrate and 3-hydroxy valerate units. In anotherembodiment, the core includes a copolymer containing 3-hydroxy butyrateand 3-hydroxy valerate units, and the shell includes a homopolymercontaining 3-hydroxy butyrate units.

Block copolymers having at least two blocks can also be prepared.Preferably, the blocks contain 3-hydroxy organoate units. For example,the first block may contain 3-hydroxy butyrate units and the secondblock may contain 3-hydroxy valerate units.

The biopolymers and biopolymer blends may be prepared usingmicroorganism hosts by controlling the nutrients available to themicroorganism, resulting in the sequential formation of differentpolymers, or polymer blocks, within the same granule. In the case ofblock copolymers, for example, the relative amounts of the nutrients andthe timing of introduction into the microorganism may be selected suchthat the first nutrient is available for reaction substantiallythroughout the process and the second nutrient is available for reactionthroughout selected portions of the process.

The biopolymers and biopolymer blends are useful in a number ofapplications. For example, they may be compounded with a tackifier and,optionally, a crosslinking agent to form an adhesive composition.Examples of suitable tackifiers and crosslinking agents are described,e.g., in Rutherford et al., U.S. Pat. No. 5,614,576 and Rutherford etal., U.S. Pat. No. 5,753,364, both of which are hereby incorporated byreference. It is also possible to combine the biopolymers and biopolymerblends with another polymer matrix to modify the properties of thepolymer matrix. For example, the two phase biopolymer particles can beincorporated into a brittle polymer such as polystyrene to improve theimpact resistance of the brittle polymer.

Other features and advantages will be apparent from the followingdescription of preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of carbon source concentration vs. time for theproduction of block copolymers from fructose and 3-hydroxy valeric acidstarting materials.

FIGS. 2A-2D are a series of microphotographs obtained from transmissionelectron microscopy throughout the course of the reaction involvingfructose and 3-hydroxy valeric acid starting materials that ultimatelyyielded polymer particles having copolymer core surrounded by ahomopolymer shell.

DETAILED DESCRIPTION

Biopolymers and biopolymer blends according to the invention arepreferably produced from microorganisms via enzyme-mediated catalysisunder nutrient-limited conditions. The microorganism serves as abioreactor. The microorganism is selected based upon the desiredbiopolymer. Specifically, microorganisms having polymerases suitable forcatalyzing the reaction of monomers to produce the desired biopolymerare selected. The polymerases may be native to the particularmicroorganism. Alternatively, the microorganism can be geneticallyengineered using known techniques to contain the desired polymerases.

One class of useful microorganisms includes bacteria. Specific examplesinclude Pseudomonas aeruginosa, Ralstonia eutropha (formerly Alcaligeneseutrophus), Rhodospirillum rubrum, and Bacillus megaterium. Each ofthese bacteria is capable of metabolizing nutrient sources includingalkanes, alkanols, organoic acids, alkenes, alkenols, alkenoic acids,and esters, for example, to produce biopolymers such as poly(hydroxyorganoates). R. eutropha is particularly useful for reacting 3-hydroxybutyric acid and 3-hydroxy organoic acid monomers having an odd numberof carbon atoms such as 3-hydroxy valeric acid, as well as precursorsthereof, to form poly(3-hydroxy organoate) polymers and copolymers.

To prepare the biopolymer or biopolymer blend, appropriate startingmaterials are introduced into the microorganism. In the case ofpoly(hydroxy organoate) biopolymers, the starting materials may includehydroxy organoic acids or precursors thereof. An example of a suitableprecursor is fructose which the microorganism metabolizes to form3-hydroxy butyric acid monomer.

The chemical composition and microstructure of the biopolymer productare influenced by the relative amounts of starting materials, theconcentration of microorganisms, and the timing of nutrient introductioninto the bioreactor (i.e., the point at which the starting material isadded to the microorganism). For example, in the case of a reactioninvolving fructose (as a source of 3-hydroxy butyric acid) and an oddchain fatty acid such as 3-hydroxy valeric acid, if fructose is fed witha limited amount of the valeric acid monomer, poly(3-hydroxy valerate)will no longer be produced after exhaustion of the valerate, resultingin a polymer particle featuring a poly(3-hydroxy (butyrate-co-valerate))copolymer core substantially surrounded by a poly(3-hydroxy butyrate)homopolymer shell. On the other hand, supplying the valeric acid monomerhalfway through particle synthesis on fructose results in a polymerparticle featuring a poly(3-hydroxy butyrate) homopolymer coresubstantially surrounded by a poly(3-hydroxy (butyrate-co-valerate))copolymer shell. By increasing cell density and/or decreasing the amountof valeric acid fed to the bacteria, it is possible to stop copolymersynthesis within a polymer chain, resulting in the formation of blockcopolymers having a poly(3-hydroxy butyrate) block and a poly(3-hydroxy(butyrate-co-valerate)) copolymer block.

A theoretical model has been developed to enable the experimentalconditions to be designed for production of a given biopolymer. Themodel (1) describes the experimentally observed molecular weightdistribution in a culture of biopolymer-producing cells, and (2)predicts transient synthesis situations encountered during the proposedpolymer synthesis process. The model can be used, for example, topredict and delineate the conditions that lead to the formation of blockcopolymers.

The model assumes that polymerase molecules sit on the surface ofpolymer granules and catalyze the addition of monomer units to thegrowing (“elongating”) polymer chain that is aggregating into or ontothe granule. After initiation, chains are elongating until anirreversible termination step detaches the chain from the polymeraseenzyme. Given a constant supply of monomer units, the polymer synthesisis governed by three rates describing initiation, elongation, andtermination of polymer chains. The model results in the followingpopulation balance that describes generally the dynamics of themolecular weight distribution of polymer chains that changes in time t:$\begin{matrix}{{{\frac{\partial{A\left( {x,t} \right)}}{\partial t} + {\frac{\partial}{\partial x}\left\lbrack {{R_{el}\left( {x,S} \right)}{A\left( {x,t} \right)}} \right\rbrack} + {{R_{t}\left( {x,S} \right)}{A\left( {x,t} \right)}}} = 0}{\frac{\partial{I\left( {x,t} \right)}}{\partial t} = {{R_{t}\left( {x,S} \right)}{I\left( {x,t} \right)}}}} & (1)\end{matrix}$

where A(x,t) and I(x,t) are the molecular weight distribution of activeand inactive chains, respectively; R_(el) is the elongation rate; R_(i)is the initiation rate; and R_(t) is the termination rate. In thegeneral case, the initiation and elongation rates depend on themolecular weight “x” of a chain and on the monomer concentration “S”.The initiation rate contributes to the physical picture through theboundary condition.

Assuming a steady state synthesis, equation (1) can be solved for theactive chain distribution on the basis of the measurable and timeinvariant overall distribution w(x), representing the sum of A(x) andI(x), using the relationship: $\begin{matrix}{{A(x)} = {\frac{1}{R_{el}} \cdot \frac{N_{A}R_{p}}{\int_{x\quad \min}^{x\quad \max}{{{xw}(x)}\quad {x}}} \cdot {\int_{x}^{x\quad \max}{{w(y)}\quad {y}}}}} & (2)\end{matrix}$

where N_(A) is Avogadro's number. After determining the active chaindistribution A(x), the termination rate R_(t) is determined using thefollowing equation: $\begin{matrix}{{R_{t}(x)} = {\frac{1}{A(x)} \cdot \frac{N_{A}R_{p}{w(x)}}{\int_{x\quad \min}^{x\quad \max}{{{xw}(x)}\quad {x}}}}} & (3)\end{matrix}$

Gel permeation chromatography can be used to measure the time invariantmolecular weight distribution for different synthesis conditions. Thisvalue can then be used to estimate the unknown active chain distributionand termination rate. Specifically, the former can be directly computedfrom the measured molecular weight distribution using Equation (2),while the latter as a function of molecular weight of chains, can beobtained using Equation (3). Once the model parameters have beenidentified, the steady state molecular weight distribution can becomputed by solving Equation (1).

This model has been used to design experiments that would produce blockcopolymers in R. eutropha using, as starting materials, fructose and3-hydroxy valeric acid. The experimental conditions predicted to yieldblock copolymers in this reaction are shown in FIG. 1. The graph in FIG.1 plots carbon source concentration vs. time. According to the graph, ifvaleric acid consumption is faster than the time required to synthesizea polymer chain, block copolymers result. The residence time of valericacid in the media can be controlled by the cell concentration and theinitial valeric acid concentration.

The invention will now be described further by way of the followingexample.

EXAMPLE

This example describes the preparation and characterization of polymergranules featuring a core containing the copolymer poly(3-hydroxy(butyrate-co-valerate)) and a shell containing poly(3-hydroxy butyrate)homopolymer. Throughout the example, “PHA” refers topoly(3-hydroxyorganoate), “PHB” refers to poly(3-hydroxy butyrate),“PHV” refers to poly(3-hydroxy valerate), and “PH(B-co-V)” refers topoly(3-hydroxy (butyrate-co-valerate)).

Materials

Epon-Araldite epoxy resin and 200-mesh copper grids for transmissionelectron microscopy (TEM) were purchased from Ted-Pella (Reading,Calif.). Homo-PHB samples were received from Monsanto. PH(B-co-V) (49%HV) was prepared in a bioreactor (conditions presented below) with R.eutropha grown on 10 g/l fructose and 1 g/l valeric acid for 24 h. Allother media components and chemicals were purchased from Sigma (St.Louis, Mo.).

Baterial Growth

R. eutropha H16 (ATCC 17699) was used in all polymer productionexperiments. Inocula were grown in 4-1 shake flasks with 1.5 l ofmineral salts medium with 30 g/l fructose and 4 g/l (NH₄)₂SO₄ to anOD₄₃₆ of 4.0 measured on a spectrophotometer (Model 8452A, HewlettPackard, Avondale, Pa.). Cells were harvested in exponential growth(μ=0.33/h) and washed twice with sterile phosphate buffer (0.036 M, pH7.0). Cells were then inoculated into 1.5 l of minimal media (˜0.4 g/l)containing 0.02 g/l (NH₄)₂SO₄ and the indicated carbon source/s (10 g/lfructose, 1 g/l valerate). Nitrogen was included to facilitateadaptation of the culture to new media conditions containing fattyacids. The low nitrogen levels could only be used to produceapproximately 6 mg/l of biomass and thus contributed insignificantly togrowth at the cell density used. Reactors were operated at 700 rpm, 30°C., pH 7 (controlled with 1M NaOH and 4% H₃PO₄), and 1 vvm air flow;dissolved oxygen remained above 90%.

Gas Chromatography (GC) analysis of PHAs

All polymer samples were analyzed by propanolysis and subsequent GCanalysis according to standard protocols described in Riis et al., J.Chromatogr. (1988) 445:285.

Substrate Analysis

Fructose levels in the media were measured using a D-Glucose/D-FructoseEnzymatic BioAnalysis kit (Boehringer Mannheim, Mannheim, Germany).Valeric acid concentrations were measured using gas chromatography (GC).GC vials were filled with 0.85 ml of sample supernatant. A total of 75μl of a 1-N HCl solution with 0.2 g/l isovaleric acid was added toacidify the samples. The isovaleric acid served as an internal standard.Samples were run on a DB-FFAP column with the following temperatureprofile: 100° C. for 5 min, ramp at 10° C./min, final temperature of200° C. for 5 min (J&W Scientific, Folsom, Calif.). Retention times wereapproximately 10.5 min for isovalerate and 11.5 min for valeric acid. Acalibration curve was established for each set of samples.

Granule Isolation

Harvested cells were concentrated, by centrifugation, approximately100-fold. The cell slurry was passed through a French Press twice at acell pressure of 10,000 psi. After settling overnight, 150 μl of settledcell slurry containing the granules was added to 15 ml of a 1% Tritonsurfactant solution at pH 13 and agitated at room temperature for 15min. Granules were centrifuged at 4000×g for 15 min and washed twicewith distilled water. Granules were dried overnight at room temperatureunder vacuum to remove all of the water.

Differential Scanning Calorimetry (DSC)

DSC was used to determine if the granules were layered structures havingtwo distinct phases. DSC analysis was carried out on a liquid nitrogencooled DSC (Pyris model, Perkin-Elmer, Norwalk, Conn.). Samples weretaken from the bioreactor at 6, 12, 18, and 24h. Granules were isolatedfrom the bacteria as described above. For glass transition measurements,9-10 mg samples were used. The temperature was ramped to 200° C., heldfor 1 min, followed by slow quenching at a rate of −20° C./min to afinal temperature of −60° C. A rate of 20° C/min from −60° C. to 30° C.was used to analyze glass transition temperatures.

TEM Preparation

TEM measurements were also used to determine if the granules werelayered structures having two distinct phases. As in the case of the DSCmeasurements, samples were taken from the bioreactor at 6, 12, 18, and24h. Dried granules were fixed in Epon-Araldite epoxy for 2 days at 60°C. The samples were cryo-microtomed at −20° C. to form a smooth face ofexposed polymer. Each epoxy block was stained with RuO₄ vapor. Specimenswere taped to the lid of a 5-ml glass vial containing 0.02 g RuCl₃-xH₂Oand 1 ml NaOCl (10-13%), sealed and stained for 7.5 h. After staining,the samples were removed and stored in a hood to dry.

Staining sufficiently hardened the samples so that cryo-microtomy was nolonger necessary. Samples were cut 70 nm thick as close to the stainedsurface as possible. Increased depth of cutting gave slices withdecreased staining, allowing a desired contrast to be achieved bychanging the cutting depth. Sections were placed on 200-mesh coppergrids and viewed in an electron microscope at 60 kV (JEM1200ex2, Joel,Boston, Mass.).

Results

PH(B-co-V) was produced during the first 12 h. of the reaction. The PHVand PHB synthesis rates were 0.030 g PHV/l per h and 0.033 g PHB/l perh, giving a copolymer composition of 48% HV. The valerate was exhaustedat approximately 12 h at a rate of 0.0894 g/l per h after which only PHBwas produced at 0.033 g PHB/l per h from the remaining fructose, whichwas utilized at a constant rate of 0.0861 g/l per h throughout all 24 hof the experiment. The concentration of PHV in the reactor remainedconstant during the last 12 h of the experiment, suggesting that nodegradation of polymer occurs concomitantly with new polymer synthesis.

DSC measurements of the isolated granules confirmed the presence of twopolymer phases in the granules. Glass transition temperatures of theindividual components PH(B-co-V) (49% HV) and PHB were determined to be−5° C. and 5° C., respectively. Each sample throughout the course of thereaction showed two distinct glass transitions, indicating the presenceof two phases. Table I shows the weight percent of each type of polymerin the granule at various stages during the course of the reaction. Allamounts are based upon percentage of total polymer weight. “% CDW”refers to the polymer percentage of cell dry weight.

TABLE I Time (h) % CDW % PHB % PH(B-co-V) 0 21 100 0 6 53 23 77 12 59 1387 18 66 31 69 24 66 41 59

The results shown in Table I show that during the first 12 h, onlyPH(B-co-V) was being produced. Nevertheless, two glass transitions wereobserved because of the PHB stored during inoculum growth, prior tointroduction into the reactor. After 12 h, only PHB is formed. The 5° C.transition was observed to increase with the increasing PHB weightfraction of the sample because glass transition heatflow changes aredirectly proportional to the mass of that component in the sample.

TEM imaging confirmed the presence of layered granules consisting of acopolymer core and a homopolymer shell. The results are shown in FIGS.2A-2D. PHB regions are shown as light gray regions, while PH(B-co-V)regions are dark gray regions.

Referring to FIGS. 2A-2D, some regions of PHB were seen in the 6 and 12h samples because of the PHB stored during inoculum growth. The granulesof PH(B-co-V) seemed to coalesce, making it difficult to discernindividual granule boundaries. The PH(B-co-V) granules likely coalescemuch easier than the PHB granules because of their lower crystallinity.At 18 and 24 h, layered granule structures became clearly visible (FIGS.2C and 2D).

Other embodiments are within the following claims.

What is claimed is:
 1. A polymer particle comprising: (a) a corecomprising a first biopolymer; and (b) a shell substantially surroundingsaid core comprising a second biopolymer different from said firstbiopolymer, said shell having a thickness that is less than about 1micrometer.
 2. A polymer particle according to claim 1 wherein saidshell has a thickness that is less than about 0.1 micrometer.
 3. Apolymer particle according to claim 1 wherein said shell has a thicknessthat is less than about 0.05 micrometer.
 4. A polymer particle accordingto claim 1 wherein said first biopolymer and said second biopolymercomprise hydroxy organoate units.
 5. A polymer particle according toclaim 4 wherein said hydroxy organoate units are selected from the groupconsisting of 3-hydroxy organoates, 4-hydroxy organoates, andcombinations thereof.
 6. A polymer particle according to claim 4 whereinsaid hydroxy organoate units are selected from the group consisting of3-hydroxy butyrate, 3-hydroxy valerate, and combinations thereof.
 7. Apolymer particle according to claim 4 wherein said first biopolymercomprises 3-hydroxy butyrate units.
 8. A polymer particle according toclaim 4 wherein said second biopolymer comprises 3-hydroxy butyrateunits.
 9. A polymer particle according to claim 4 wherein said firstbiopolymer comprises 3-hydroxy valerate units.
 10. A polymer particleaccording to claim 4 wherein said second biopolymer comprises 3-hydroxyvalerate units.
 11. A polymer particle according to claim 1 wherein saidfirst biopolymer is a homopolymer and said second biopolymer is acopolymer.
 12. A polymer according to claim 11 wherein said secondbiopolymer is a block copolymer.
 13. A polymer particle according toclaim 11 wherein said first biopolymer is a homopolymer comprising3-hydroxy butyrate units and said second biopolymer is a copolymercomprising 3-hydroxy butyrate and 3-hydroxy valerate units.
 14. Apolymer particle according to claim 1 wherein said first biopolymer is acopolymer and said second biopolymer is a homopolymer.
 15. A polymerparticle according to claim 14 wherein said first biopolymer is a blockcopolymer.
 16. A polymer particle according to claim 14 wherein saidfirst biopolymer is a copolymer comprising 3-hydroxy butyrate and3-hydroxy valerate units, and said second biopolymer is a homopolymercomprising 3-hydroxy butyrate units.
 17. A polymer particle comprising:(a) a core comprising a first biopolymer comprising units selected fromthe group consisting of 3-hydroxy butyrate, 3-hydroxy valerate, andcombinations thereof; and (b) a shell substantially surrounding saidcore comprising a second biopolymer comprising units selected from thegroup consisting of 3-hydroxy butyrate, 3-hydroxy valerate, andcombinations thereof, wherein said second biopolymer is different fromsaid first biopolymer.
 18. A polymer particle according to claim 17wherein said first biopolymer is a homopolymer and said secondbiopolymer is a copolymer.
 19. A polymer particle according to claim 18wherein said first biopolymer is a homopolymer comprising 3-hydroxybutyrate units and said second biopolymer is a copolymer comprising3-hydroxy butyrate and 3-hydroxy valerate units.
 20. A polymer particleaccording to claim 17 wherein said first biopolymer is a copolymer andsaid second biopolymer is a homopolymer.
 21. A polymer particleaccording to claim 20 wherein said first biopolymer is a copolymercomprising 3-hydroxy butyrate and 3-hydroxy valerate units, and saidsecond biopolymer is a homopolymer comprising 3-hydroxy butyrate units.22. A process for preparing a polymer particle comprising introducingfirst and second nutrients into a microorganism comprising a polymerasecapable of processing said nutrients to form biopolymers, wherein therelative amounts of said nutrients and the timing of introduction intosaid microorganism are selected to produce a polymer particlecomprising: (a) a core comprising a first biopolymer; and (b) a shellsubstantially surrounding said core comprising a second biopolymerdifferent from said first biopolymer, said shell having a thickness thatis less than about 1 micrometer.
 23. A process according to claim 22wherein said microorganism comprises a bacterium.
 24. A processaccording to claim 22 wherein said bacterium comprises R. eutropha. 25.A process according to claim 22 wherein said first nutrient comprises ahydroxy organoic acid, or a precursor thereof, and said second nutrientcomprises a hydroxy organoic acid, or a precursor thereof, that isdifferent from said first nutrient.
 26. A process according to claim 25wherein said first nutrient comprises 25 3-hydroxy butyric acid, or aprecursor thereof, and said second nutrient comprises 3-hydroxy valericacid, or a precursor thereof.
 27. A process according to claim 26wherein said first nutrient comprises fructose.
 28. A process accordingto claim 22 wherein the relative amounts of said nutrients and thetiming of introduction into said microorganism are selected to produce apolymer particle comprising: (a) a core comprising a first biopolymer inthe form of a homopolymer; and (b) a shell substantially surroundingsaid core comprising a second biopolymer in the form of a copolymer. 29.A process according to claim 28 wherein said first nutrient comprises ahydroxy organoic acid, or a precursor thereof, and said second nutrientcomprises a hydroxy organoic acid, or a precursor thereof, that isdifferent from said first nutrient.
 30. A process according to claim 29wherein said first nutrient comprises 3-hydroxy butyric acid, or aprecursor thereof, and said second nutrient comprises 3-hydroxy valericacid, or a precursor thereof, said polymer particle comprising: (a) acore comprising a homopolymer comprising 3-hydroxy butyrate units; and(b) a shell substantially surrounding said core comprising a copolymercomprising 3-hydroxy butyrate and 3-hydroxy valerate units.
 31. Aprocess according to claim 22 wherein the relative amounts of saidnutrients and the timing of introduction into said microorganism areselected to produce a polymer particle comprising: (a) a core comprisinga first biopolymer in the form of a copolymer; and (b) a shellsubstantially surrounding said core comprising a second biopolymer inthe form of a homopolymer.
 32. A process according to claim 31 whereinsaid first nutrient comprises a hydroxy organoic acid, or a precursorthereof, and said second nutrient comprises a hydroxy organoic acid, ora precursor thereof, that is different from said first nutrient.
 33. Anadhesive composition comprising: (A) a plurality of polymer particles,each of which comprises: (a) a core comprising a first biopolymer; and(b) a shell substantially surrounding said core comprising a secondbiopolymer different from said first biopolymer, said shell having athickness that is less than about 1 micrometer; and (B) a tackifier. 34.An adhesive composition according to claim 33 wherein said compositionis crosslinked.